The present invention relates to Lyme Disease (Borrelia burgdorferi antigen) compositions, especially combination compositions, and to methods of making and using the same, especially for veterinary uses. The compositions can include, in addition to a Borrelia burgdorferi antigen or antigens, an antigen for an additional pathogen, such as a canine, feline or equine pathogen, for instance an antigen from at least one of: rabies virus, canine distemper virus, adenovirus, corona virus, parainfluenza, parvovirus, FeLV, feline herpesvirus, equine influenza virus, equine herpes virus, and the like. The compositions advantageously induce an immunological response against Lyme Disease (Borrelia burgdorferi) infections, as well as against any other antigen in the composition, when administered to a host. The compositions elicit long-term immunity (response) against Lyme Disease Borrelia burgdorferi in animals, including horses and dogs, and afford protection or elicit immunological response in the animals. In combination compositions, there is an absence of efficacy interference.
The invention further relates to methods for making and using such compositions.
The invention additionally relates to the antibodies elicited by the compositions, isolated from an animal or cell culture as the case may be, which are useful for preparing a diagnostic kit, test or assay for the detection of a Borrelia burgdorferi antigen or Lyme Disease or another antigen of another pathogen or another pathogen.
Lyme disease is a multisystem illness, transmitted by ticks of the Ixodes ricinus complex. The spirochaete Borrelia burgdorferi sensu lato is the aetiologic agent of Lyme disease, which is now the most common arthropod borne disease in the United States, and is endemic in Central Europe (Barbour et al., 1993). Although curable by antibiotic therapy in its early stages, if Lyme disease is allowed to progress, cardiac, neurological and joint abnormalities can arise. Investigations into the development of a human vaccine for Lyme disease are under way. The outer surface lipoprotein OspA of Borrelia burgdorferi is the current major candidate molecule for development of such a vaccine. Recombinant OspA lipoprotein (rOspA) is known to elicit a protective immune response in mice against challenge by infectious B. burgdorferi (Fikrig et al., 1990; Erdile et al., 1993; U.S. Ser. No. 08/373,455). OspA is currently undergoing human field trials as a subcutaneously administered vaccine in the United States (Keller et al., 1994).
Above-cited applications WO93/08299 and PCT/US92/08697 relate to recombinant OspA (rOspA) vaccines, especially lipidated rOspA, and methods for expressing DNA encoding OspA, and isolating the lipidated rOspA. Above-cited U.S. Pat. Nos. 5,582,990 and 5,523,089 and application WO 90/04411 relate to DNA encoding OspA, the amino acid sequence of OspA including rOspA and lipidated forms thereof, synthetic OspA including rOspA and lipidated forms thereof, compositions containing OspA or synthetic OspA, and methods of using such compositions. And, the other above-cited applications relate to DNA encoding other Borrelia antigens or other Osps, or to DNA encoding useful fragments of OspA or of other Osps, amino acid sequences thereof, compositions containing such fragments or other Osps, and methods for using such compositions. DNA from documents cited herein pertaining to Borrelia burgdorferi can be used in the methods of U.S. Pat. Nos. 5,582,990, and 5,523,089 or PCT/US92/08697 to produce OspA, other Borrelia antigens or Osps, or fragments thereof, for use in this invention. In regard to DNA and antigens useful in this invention, reference is also made to Molecular Microbiology (1989), 3(4), 479-486.
A particular problem in the art involves the infection of domesticated animals with Borrelia burgdorferi from tick bites. For instance, dogs and horses are susceptible to Lyme Disease due to tick bites, and their masters are unaware of the infection until it is too late (the tell-tale circular ring around the tick bite being undetected due to fur, and the dog or horse is unable to verbalize complaints such as sore joints, etc. from the infection, or due to masters not appreciating the subtle symptoms of the disease in animals). In addition, there is a concern about possible transmission to humans.
A further problem in the art involves vaccination strategies. More specifically, when vaccinating domesticated animals it is preferred to administer multiple antigens in one, xe2x80x9ccocktailxe2x80x9d or multivalent composition; for instance, to reduce the number of shots and number of visits to the veterinarian.
A Lyme Disease combination or xe2x80x9ccocktailxe2x80x9d or multivalent vaccine or immunological or immunogenic composition (Borrelia burgdorferi antigen in combination with other antigens in a composition, particularly for canines), is not presently available or known.
A still further problem in the art, especially as to multivalent composition, involves xe2x80x9cefficacy interferencexe2x80x9d, namely a failure of one or more antigens, in a combination composition to maintain or achieve efficacy. This is believed due to interference on that antigen stimulating an immunological, antigenic, antibody, or protective response in the host, e.g., dog, when administered, because of the presence of the other antigens. For instance, rabies antigens in a combination with other antigens suffer interference from or interfere with the stimulation of an immunological, antigenic, antibody or protective response by those other antigens in such a composition, especially when that composition is administered to dogs. More particularly, antigens, such as rabies antigens and Leptospira antigens, when administered with one or more other antigens can interfere with the response elicited by those antigens. Indeed, Leptospira antigens can interfere with OspA. However, for other hosts, such as cats, combination vaccines are known. Perhaps, without wishing to be bound by any one theory, the xe2x80x9cefficacy interferencexe2x80x9d is due to some peculiarity of the canine biological system or, to the reaction with the canine biological system by presently known antigens or, by the combination thereof.
Regardless of the theory, there is heretofore to the inventor""s knowledge, no known Lyme Disease combination with other antigenic composition, especially for canine use, and which does not exhibit efficacy interference. There is a need for a Lyme Disease combination, especially for canine use. It would indeed be surprising, unexpected and non-obvious to be able to formulate a Lyme Disease combination (with other antigens) composition which exhibits a lack of efficacy interference in canines, especially because as shown by the present knowledge and efficacy interference, one cannot simply combine xe2x80x9cantigen compositionsxe2x80x9d to prepare a useful combination or xe2x80x9ccocktailxe2x80x9d composition.
Additionally, it would be advantageous if such a Lyme Disease antigen cocktail composition provided long term protection for canines, as well as protection for pups with maternal immunity to Lyme Disease. As the skilled artisan is aware, maternal immunity is immunity that a newborn acquires from its mother upon birth and/or from nursing, which immunity, after a period of time, lapses in the newborn, thereby leaving the newborn susceptible. Furthermore, the presence of maternal antibodies in the newborn prevents the newborn from obtaining a protective response when administered an antigen composition, e.g., a vaccine, thus meaning that the newborn must enter a period of no or little immunity, i.e., susceptibility, to the danger of the newborn before administration of an antigen or vaccine composition can be considered. In regard to maternal immunity, reference is made to U.S. Pat. No. 5,338,683, issued Aug. 16, 1994 and incorporated herein by reference.
Thus, alternative vaccination strategies are desirable.
It would even be more advantageous, surprising and unexpected if the Borrelia burgdorferi antigen which can be used in a combination xe2x80x9ccocktailxe2x80x9d composition which lacks efficacy interference in canines, can be used in such a composition for other hosts such as felines, equines and the like and, which provides long-term protection in dogs as well as protection in pups in spite of maternal immunity, was a recombinant antigen.
In particular, it is believed that heretofore the art has not taught or suggested administration to a mammalian hostxe2x80x94especially a domesticated animal such as dogs, cats or horsesxe2x80x94susceptible to Lyme disease, of a combination composition including a Borrelia burgdorferi antigen, e.g., OspA, especially as herein disclosed.
Immunization with a monovalent Lyme Disease (Borrelia burgdorferi) vaccine (designated Ly) is shown to be safe and efficacious in dogs, as assessed by both rate of seroconversion and protection against Borrelia burgdorferi infection.
Studies have now shown that a Lyme vaccine, containing 10 xcexcg/ml OspA (prepared according to WO93/08299 and U.S. Pat. Nos. 5,582,990 and 5,523,089, incorporated herein by reference), provides dogs with significant protection against tick challenge, as assessed by both a reduction in spirochete proliferation and prevention of clinical disease. Furthermore this vaccine-induced immunity is still significant when challenge is five to six months after vaccination. The monovalent vaccine has also been shown to be exceptionally safe; dogs displaying clinical signs of Lyme Disease show no exacerbation of disease when vaccinated with repeated, high doses of the OspA vaccine.
However, it would be an even more significant advance in the art to provide a safe and efficacious combination Lyme vaccine. It would be a more significant advance to have lack of interference of the OspA vaccine when administered with other canine antigens. The herein results show that subcutaneous vaccinations, given at intervals, result in significant seroconversion. The level of antibody induced in these vaccinates is similar to that observed in dogs receiving the monovalent vaccine; levels which were shown to protect vaccinates including long termxe2x80x94against a challenge using Borrelia-infected ticks.
Because there is evidence that horses are also susceptible naturally to Borrelia burgdorferi infection, it would also be a significant advance to provide equine Lyme vaccines which result in the production of significant Borrelia burgdorferi antigen, e.g., OspA, antibody in horses.
It is an object of the present invention to provide a combination Borrelia burgdorferi, e.g., OspA, vaccine or immunological or antigenic composition, especially such a composition which induces protection in dogs, horses, or other domesticated animals.
It is another object of the present invention to provide a combination Lyme Disease vaccine or immunological or antigenic composition wherein there is no significant efficacy interference by the antigens in the combination.
It is a further object of the present invention to provide a Lyme Disease vaccine or immunological or antigenic composition for producing a serological response upon administration to dogs, horses or other domesticated animals.
Accordingly, the present invention provides an immunological antigenic or vaccine composition comprising an isolated purified Borrelia burgdorferi antigen, at least one additional antigen of a mammalian pathogen other than Borrelia burgdorferi, and optionally a pharmaceutically or veterinarily acceptable carrier.
In the composition, the isolated, purified Borrelia burgdorferi antigen can comprise isolated, purified OspA.
Further still, in the composition, the isolated, purified OspA can be an isolated, purified, lipidated recombinant OspA which is substantially free of lipopolysaccharide and substantially free of other bacterial proteins.
The composition can be without any immunogenicity enhancing adjuvant.
In the composition, the additional antigen can be selected from the group consisting of: an antigen of a canine pathogen, an antigen of an equine pathogen, and an antigen of a feline pathogen.
In certain preferred embodiments, in the composition, the additional antigen is an antigen of a canine pathogen or of an equine pathogen.
The additional antigen can be selected from the group consisting of: a rabies virus antigen, a canine distemper antigen, an adenovirus antigen, a coronavirus antigen, a parainfluenza antigen, a parvovirus antigen, and mixtures thereof.
In certain preferred embodiments of the composition, the additional antigen is a rabies virus antigen; or, the additional antigen comprises a canine distemper antigen, an adenovirus antigen, a coronavirus antigen, a parainfluenza antigen, and a parvovirus antigen.
The additional antigen can be a modified or attenuated live virus, e.g., an attenuated CDV, CAV such as CAV2, a coronavirus, a parainfluenza virus and/or a parvovirus.
The invention further comprehends a method for eliciting an immunological response in a mammal susceptible to Lyme Disease and to the mammalian pathogen other than Borrelia burgdorferi comprising administering to the mammal the aforementioned compositions.
The mammal can also be a horse and the additional antigen is of an equine pathogen. The mammal can also be a dog or pup and the additional antigen is of a canine pathogen.
Still further, the invention provides a method for eliciting an immunological response in a horse against Borrelia burgdorferi comprising administering to the horse a composition comprising isolated purified Borrelia burgdorferi OspA.
Even further, the invention provides a method for eliciting an immunological response in a dog or pup against Borrelia burgdorferi comprising administering to the dog or pup a composition comprising isolated, purified Borrelia burgdorferi OspA.
In these methods the OspA can be an isolated, purified lipidated recombinant OspA which is substantially free of lipopolysaccharide, and substantially free of other bacterial proteins. And, in these methods, the composition can be without any immunogenicity-enhancing adjuvant.
The invention also comprehends a method for preparing the aforementioned compositions comprising preparing the additional antigen in lyophilized form, preparing the Borrelia burgdorferi antigen in liquid form, and rehydrating the additional antigen with the Borrelia burgdorferi antigen.
The Borrelia burgdorferi OspA can be obtained by transforming a host organism by a plasmid containing a gene coding for a full-length wild-type Borrelia burgdorferi OspA lipoprotein and producing recombinant Borrelia burgdorferi OspA lipoprotein, and purifying said recombinant Borrelia burgdorferi OspA lipoprotein substantially free from other bacterial protein and from lipopolysaccharide under non-denaturing conditions from a lysate of said host organism.
For instance, useful in this invention is an isolated lipoprotein which comprises purified recombinant Borrelia burgdorferi OspA lipoprotein which has retained lipidation, is substantially free from other bacterial proteins and is substantially free from lipopolysaccharide, and the purified recombinant Borrelia burgdorferi OspA lipoprotein having been obtained by a process which comprises:
transforming a host organism by a plasmid containing a gene coding for a full-length wild-type Borrelia burgdorferi OspA lipoprotein and producing recombinant Borrelia burgdorferi OspA lipoprotein,
purifying the recombinant Borrelia burgdorferi OspA lipoprotein substantially free from other bacterial protein and from lipopolysaccharide under non-denaturing conditions from a lysate of said host organism so as to obtain a purified recombinant Borrelia burgdorferi lipoprotein which remains lipidated and is immunogenic to a mammalian host when administered to the mammalian host.
The purifying of the recombinant Borrelia burgdorferi OspA can be by:
lysing cells of the host organism to obtain lysed cells;
treating the lysed cells with a surfactant which selectively solubilizes Borrelia burgdorferi OspA lipoprotein in preference to bacterial and other proteins and which is able to effect phase separation of a detergent phase under mild temperature conditions of about 35xc2x0 to 40xc2x0 C., to obtain treated lysed cells;
separating by phase separation the treated lysed cells into a detergent phase containing solubilized Borrelia burgdorferi OspA lipoprotein, an aqueous phase containing bacterial and other proteins and a solid phase containing cell residue;
separating the detergent phase from the solid and aqueous phases;
contacting the detergent phase with a chromatography column under conditions which result in binding of proteins other than Borrelia burgdorferi OspA lipoprotein to the chromatography column; and
recovering flow-through from the first chromatography column containing the Borrelia burgdorferi OspA lipoprotein freed from the bound proteins.
The purifying of the recombinant Borrelia burgdorferi OspA can be by the contacting of the detergent phase with the chromatography column at a pH of about 7.5.
Alternatively, the OspA can be obtained by a process for the production of an isolated and purified recombinant OspA lipoprotein encoded by a full-length wild-type Borrelia opsa gene, which comprises:
effecting induction of Borrelia ospa lipoprotein from a host organism transformed by a plasmid containing the ospA gene,
lysing the cells of the host organism,
treating the lysed cells with a surfactant which selectively solubilizes Borrelia OspA lipoprotein in preference to bacterial and other proteins and which is able to effect phase separation of a detergent phase under mild conditions,
effecting phase separation into a detergent phase containing solubilized Borrelia OspA lipoprotein, an aqueous phase containing bacterial and other proteins and a solid phase containing cell residue,
separating the detergent phase from the solid phase and the aqueous phase, and
purifying the detergent phase free from proteins other than Borrelia Ospa lipoprotein and lipopolysaccharide by:
(a) contacting the detergent phase with a first chromatography column under conditions which result in binding of proteins other than the Borrelia OspA lipoprotein to the first chromatography column,
(b) recovering the flow through from the first chromatography column containing the Borrelia OspA lipoprotein freed from the bound proteins,
(c) contacting the flow through from the first chromatography column with a second chromatography column under conditions to result in binding of the Borrelia OspA lipoprotein to the second chromatography column in preference to any residual contaminating proteins and lipopolysaccharide which flow through the second chromatography column,
(d) contacting the second chromatography column with an eluant under conditions to elute the bound Borrelia OspA lipoprotein from the second chromatography column, and
(e) collecting elute containing Borrelia OspA from said second chromatography.
Still further, in this process the contacting of the flow through with the second chromatography column can be effected at a pH of below about 5.7 and effective to bind the Borrelia OspA lipoprotein to the second chromatography column with an eluant is effected at a pH of up to and above about 5.7 and effective to elute bound Borrelia OspA lipoprotein from the second chromatography column.
And, even further still in this process the contacting of the detergent phase with the first chromatography column can be effected at a pH of approximately 7.5, the contacting of the flow through with the second chromatography column can be effected at a pH of approximately 4.2 and the contacting of the second chromatography column with an eluant can be effected at a pH of approximately 5.7.
The invention further comprehends antibodies elicited by the compositions and methods.
Surprisingly, no significant efficacy interference is observed from the inventive compositions and methods.
Other Borrelia antigens, for use in the compositions, methods and processes aforementioned in addition or in the alternative to OspA, are inter alia, as discussed in the applications and documents cited therein set forth under xe2x80x9cRelated Applications,xe2x80x9d including methods for preparing those other antigens.
Other objects and embodiments are disclosed in or are obvious from the following Detailed Description.